Vaccine Composition for Treating or Preventing Shigellosis

ABSTRACT

The present invention relates to a  Shigella  strain, of which the surface exposure of a protective antigen existing on a cellular membrane is increased due to the destruction of a wzy gene of  Shigella  sp., and to a vaccine composition for treating or preventing shigellosis, containing the mutant  Shigella  strain as an active ingredient. The  Shigella  strain of the present invention has a cell wall with a reduced thickness since a gene encoding a protein necessary for polymerization of the O-saccharide antigen is deleted, and as a result, membrane antigens including protein antigens commonly existing in different  Shigella  spp. are more exposed to immune cells, and thus the  Shigella  strain can be favorably used as a vaccine composition for treating or preventing shigellosis, derived from various  Shigella  spp.

TECHNICAL FIELD

The present invention relates to a vaccine composition for treating orpreventing shigellosis, and more particularly, to a Shigella straincapable of being effectively used in a vaccine composition forpreventing shigellosis derived from various Shigella species because thesurface exposure of a protective antigen existing on a cellular membraneincreases due to the destruction of a wzy gene of Shigella species, anda vaccine composition for treating or preventing shigellosis includingthe same.

BACKGROUND ART

Shigella sp. is a Gram-negative bacterial pathogen that causesshigellosis in humans by infecting epithelial cells of the colon.Shigella primarily infects intestinal epithelial cells, expressesseveral proteins which provide a mechanism for delivering effectorsinducing the bacterial uptake into host cells via phagocytosis. Toaccomplish the injection of the effectors, Shigella uses a type IIIsecretion (TTS) system to induce the entry into epithelial cells andtrigger apoptosis in infected macrophages.

Bacteria of Shigella sp., including Shigella dysenteriae (S.dysenteriae), Shigella flexneri (S. flexneri), Shigella boydii (S.boydii), and Shigella sonnei (S. sonnei), are responsible forshigellosis in humans, a disease characterized by the destruction of thecolonic epithelium that is responsible for 1 million deaths per year indeveloping countries. Shigella dysenteriae has 15 serotypes, Shigellaflexneri has 14 serotypes and subtypes, Shigella boydii has 20serotypes, and Shigella sonnei has one serotype, but the prevalence ofthese strains is not evenly distributed.

Although it is possible to control and treat shigellosis outbreaks withantibiotics, the high cost of antibiotics and the constant emergence ofantibiotic-resistant Shigella species, even against to the newestantibiotics, underscore a need for effective vaccines to help controlShigella and related enteroinvasive E. coli diseases in the developingregions of the world.

Natural Shigella infections confer immunity and provide protectionagainst subsequent infections with homologous virulent Shigella.Epidemiologic and volunteer studies have revealed that protectiveimmunity against Shigella is directed against the LPS or O-specificantigens, and thus is associated with serotypes of Shigella. Manystudies have been conducted for Shigella vaccines including the use oflive attenuated Shigella, dead Shigella whole bacteria, and Shigellalipopolyssacharides (LPSs) or O-polysaccharides conjugated to carrierssuch as proteosomes, tetanus toxoids, and ribosomes. Despite severalyears of extensive research, however, any effective and inexpensivevaccines against such Shigella species are not yet available.

When the attenuated Shigella strains are used as live oral vaccines, ithas been demonstrated to induce protective efficacy. The results ofclinical trials of genetically well characterized invasive Shigellavaccines are promising. It has been demonstrated orally administeredCVD1208, SC602, WRSS1, and WRSd1 vaccines are safe and immunogenic involunteer trials, and particularly that SC602 protects againstshigellosis. Clinical trials using CVD1208 demonstrated that thesymptoms of mild fever and diarrhea, which are observed when using someof the live Shigella vaccines, may be reduced by elimination of sen andset genes from the vaccine strains. Studies on Shigella diarrhea in sixAsian countries indicated that a relative distribution of Shigellaspecies isolated from patients varies for different countries and sites.Moreover, the Shigella flexneri serotypes are highly heterogeneous in adistribution thereof from site to site, and even from year to year. Theheterogeneous distribution of Shigella species and serotypes suggestthat multivalent or cross-protective Shigella vaccines will be requiredto prevent shigellosis all over the world. Vaccines that aim to confer awide spectrum of coverage may need to include all important Shigellaserotypes. To solve such a dilemma, a vaccine strategy based on the useof ‘pentavalent formulations’ including the attenuated Shigella sonneiand Shigella dysenteriae 1 strains along with Shigella flexneri 2a, 3aand 6 strains has been advocated. On the other hand, the use of complexstructures consisting of Shigella-derived serotype-specific andcross-reactive antigens such as whole dead or live attenuated bacteriahas, for example, been considered to be a promising approach tovaccinate against infections caused by the most common species andserotypes of Shigella (WO 2010/046778 A2).

Meanwhile, thick O-polysaccharides exist on a cell wall due to an actionof a wzy enzyme (O-antigen polymerase) in the case of the Shigellaspecies, and thus various protein antigens existing on a cellularmembrane and common or specific to the species are buried in a cell wallto block exposure to immune cells. Therefore, such protein antigens havemany limitations in use for immunity against Shigella.

DISCLOSURE Technical Problem

Therefore, it is an aspect of the present invention to provide agenetically engineered Shigella strain in which surface exposure of across-protective antigen increases, and a vaccine composition fortreating or preventing shigellosis using the Shigella strain.

Technical Solution

To solve the above problems, the present invention provides a Shigellastrain in which surface exposure of a protective antigen existing on acellular membrane increases due to the destruction of a wzy gene ofShigella species.

According to one exemplary embodiment of the present invention, the wzygene may have one base sequence selected from the group consisting ofSEQ ID NOs: 1 to 5, but the present invention is not limited thereto.

According to another exemplary embodiment of the present invention, theprotective antigen may be an IcsP2 or SigA2 protein, but the presentinvention is not limited thereto. According to preferred exemplaryembodiments of the present invention, the IcsP2 protein may have anamino acid sequence set forth in SEQ ID NO: 6, and the SigA2 protein mayhave an amino acid sequence set forth in SEQ ID NO: 7, but the presentinvention is not limited thereto.

According to one exemplary embodiment of the present invention, theShigella sp. may be selected from the group consisting of Shigelladysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei.According to preferred exemplary embodiments of the present invention,the Shigella sp. may be selected from the group consisting of Shigelladysenteriae type 1, Shigella dysenteriae 2, Shigella flexneri 2a,Shigella flexneri 3a, Shigella flexneri 5a, Shigella flexneri 5b,Shigella flexneri 6, Shigella boydii serotype 4, Shigella boydii 7, andShigella sonnei 482-79, but the present invention is not limitedthereto.

Also, the present invention provides a vaccine composition for treatingor preventing shigellosis, which includes the aforementioned Shigellastrain in which surface exposure of a protective antigen existing on acellular membrane increases due to the destruction of a wzy gene ofShigella sp.

According to one exemplary embodiment of the present invention, theShigella strain may be an attenuated strain that may be selected fromthe group consisting of a live strain and a dead strain.

According to another exemplary embodiment of the present invention, thevaccine composition may further include an adjuvant, and the adjuvantmay be selected from the group consisting of an aluminum salt, an immunestimulating complex (ISCOM), a saponin-based adjuvant, an oil-in-wateremulsion, a water-in-oil emulsion, a toll-like receptor ligand such as amuramyl dipeptide, E. coli LPS, an oligonucleotide containingunmethylated DNA, poly(I:C), lipoteichoic acid, a peptidoglycan, acholera toxin, a heat-labile E. coli enterotoxin, a pertussis toxin, anda Shiga toxin, but the present invention is not limited thereto.

According to one exemplary embodiment of the present invention, thevaccine composition may be administered by injection or via a mucosalroute, but the present invention is not limited thereto. According topreferred exemplary embodiments of the present invention, the vaccinecomposition may be administered by subcutaneous, intradermal, orintramuscular injection, and the mucosal route may be selected from thegroup consisting of oral, buccal, sublingual, intranasal, and rectal,but the present invention is not limited thereto.

According to one exemplary embodiment of the present invention, aneffective dose of the vaccine composition may be in a range ofapproximately 10 μg to approximately 2 mg, but the present invention isnot limited thereto.

Advantageous Effects

The Shigella strain of the present invention has a cell wall with areduced thickness because a gene encoding a protein required forpolymerization of an O-saccharide antigen is deleted. Therefore becausemembrane antigens including protein antigens commonly existing indifferent Shigella species are more exposed to immune cells, theShigella strain can be effectively used as a vaccine composition fortreating or preventing shigellosis derived from various Shigella sp.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram schematically showing a cell wall structure of awild-type Shigella strain and a ΔWZY Shigella strain according to thepresent invention.

FIG. 2 shows results of lipopolyssacharide (LPS) analysis using silverstaining. LPSs derived from the wild-type Shigella and ΔWZY strains areanalyzed by 14% tris/tricine SDS PAGE (30 ng/lane; 16.5% gel and 100ng/lane in the case of Shigella flexneri 2a WT and ΔWZY) and silverstaining. A ladder pattern specific to the LPS (O-antigenpolymerization) is observed from the wild type, but such a patterndisappears from the ΔWZY.

FIGS. 3A to 3C are graphs illustrating survival rates of laboratoryanimals after challenges. Mice are intranasally immunized with ΔWZYthree times at intervals of 2 weeks. One week after the lastimmunization, the mice are intranasally challenged with Shigellaflexneri 2a or 6 and Shigella dysenteriae. The survival of the animalsis monitored daily. * represents a value of p<0.05 using a log-ranktest.

FIGS. 4A to 4C are graphs illustrating that ΔWZY provides protectionagainst homologous challenges of Shiglla in mice. Mice are intranasallyimmunized with live ΔWZY, formalin-inactivated ΔWZY (FI ΔWZY), or eachof positive strains (Shigella flexneri 2a-SC602; Shigella flexneri6-0.13% FI wild type; and Shigella sonnei-4% FI wild type) three timesat intervals of 2 weeks. One week after the last immunization, the miceare intranasally challenged with Shigella flexneri 2a 2457T or 6, andShigella sonnei. The survival of the animals is monitored daily. Five tosix mice are used per each group (N=5 to 6).

FIGS. 5A and 5B are graphs illustrating results of survival tests forheterologous protection. Mice are intranasally immunized with live ΔWZY,formalin-inactivated ΔWZY (FI ΔWZY), or each of positive strains(Shigella flexneri 2a-SC602; Shigella flexneri 6-0.13% FI wild type; andShigella sonnei-4% FI wild type) three times at intervals of 2 weeks.One week after the last immunization, the mice are intranasallychallenged with Shigella flexneri 2a 2457T or 6. The survival of theanimals is monitored daily. Five mice are used per each group (N=5).

FIGS. 6A and 6B are graph illustrating that ΔWZY immunization in miceinduces systemic humoral and local immune responses. Mice areintranasally immunized with 5 μg of each of live SC602, live Shigellaflexneri 2a (ΔWZY), Shigella flexneri 2a (0.13% FIΔWZY), and Shigellaflexneri 2a (0.13% FIΔWZY+dmLT) 3 times at intervals of 2 weeks. Sixdays after the third immunization, a serum is collected from theindividual mice and IgG is detected by ELISA. * represents a value ofp<0.05 using a t-test (n=4 to 5).

FIGS. 7A to 7E are graph illustrating results of flow cytometricanalysis of expression of PSSP on bacteria (using an anti-PSSP-1(=IcsP2) antibody). A larger amount of IcsP is detected in ΔWZY,compared to wild-type Shigella. It is assumed that an outer membraneprotein, IcsP, is buried by O-polysaccharides in the wild-type strainbut easily detected on a surface of the ΔWZY due to a shorter length ofone unit O-antigen.

BEST MODE

The present invention provides a Shigella strain in which surfaceexposure of a protective antigen existing on a cellular membraneincreases due to the destruction of a wzy gene of Shigella species, anda vaccine composition for treating or preventing shigellosis, whichincludes the Shigella strain.

According to one exemplary embodiment of the present invention, anattenuated Shigella strain (hereinafter referred to as ‘ΔWZY’) isprepared by destructing a wzy (O-antigen polymerase) gene. According topreferred exemplary embodiments of the present invention, an attenuatedShigella flexneri (S. flexneri) 2a strain is prepared by destructing awzy (O-antigen polymerase) gene, and the resulting Shigella flexneri 2aΔWZY expresses only one unit O-antigen, and thus has a cell wall with aremarkably reduced thickness, compared to the wild-type strain (see FIG.1). According to another exemplary embodiment of the present invention,the ΔWZY strain shows increased exposure of surface protein antigens,compared to the native strain.

According to preferred exemplary embodiments of the present invention,the Shigella strain of the present invention has polysaccharides on acell wall with a reduced thickness, and thus shows an increased surfaceexposure of potentially protective antigens which are buried in the cellwall of the wild-type strain. Therefore, an immunization strategy usingthe mutant strain whose cell wall has a reduced thickness due to thedestruction of the wzy gene as described above may be effectivelyapplied to immunization with other gram-negative bacteria as well as theShigella sp.

According to one exemplary embodiment of the present invention, when theShigella flexneri 2a ΔWZY is intranasally administered to mice, the miceare protected against experimental lung pneumonia induced by strainsbelonging to different species and serotypes, including Shigellaflexneri 2a, Shigella flexneri 6, and Shigella dysenteriae (S.dysenteriae) 1.

According to one exemplary embodiment of the present invention, otherΔWZY strains of Shigella species such as Shigella sonnei 482-79(pWR105), Shigella dysenteriae 2, and Shigella flexneri 6 ΔWZY areprepared, and animal experiments are planned for cross-protectionagainst Shigella challenges, followed by ΔWZY immunization with orwithout an adjuvant (dmLT) (E. B. Norton, L. B. Lawson, L. C. Freytag,J. D. Clements, Characterization of a mutant Escherichia coliheat-labile toxin, LT (R192G/L211A), as a safe and effective oraladjuvant, Clin Vaccine Immunol 18 (2011) 546-551).

According to preferred exemplary embodiments of the present invention,preventive vaccination with the genetically modified Shigella strain ofthe present invention with an enhanced exposure of common outer membraneproteins may be used alone or in combination with adjuvant as onecomponent of a Shigella vaccine formulation.

According to one exemplary embodiment of the present invention, theShigella strain disclosed in the present invention may be administeredtogether with a pharmaceutically acceptable diluent. Such a formulationmay be administered by injection (subcutaneous, intradermal, orintramuscular), or may be locally applied onto the skin using anadhesive patch. On the other hand, the vaccine may be administered via amucosal route (oral, buccal, sublingual, intranasal, nasal drops,rectal) using a pharmaceutically acceptable vehicle. The Shigella strainmay also be mixed with an adjuvant to improve the ensuing immuneresponses. Examples of such an adjuvant may include an aluminum salt,ISCOM, a saponin-based adjuvant, oil-in-water and water-in-oilemulsions, a toll-like receptor ligand such as a muramyl dipeptide, E.coli LPS, an oligonucleotide containing unmethylated DNA, poly(I:C),lipoteichoic acid, and a peptidoglycan, but the present invention is notlimited thereto. Active derivatives such as a cholera toxin, aheat-labile E. coli enterotoxin, a pertussis toxin, a Shiga toxin, andan analogue may be used as the enterotoxin and adjuvant thereof.

According to the present invention, various means and techniques,including what are generally used in molecular immunology, cellularimmunology, pharmacology and microbiology, may fall within the technicalsprits and scopes of the present invention. Such means and techniquesare specifically disclosed in various prior-art documents known to aperson having ordinary skill in the art. The abbreviations disclosed inthis specification correspond to units of measure, techniques,properties, or compounds as follows: “min” represents minute, “h”represents hour(s), “μL” represents microliter(s), “mL” representsmilliliter(s), “mM” represents millimole, “M” represents mole, “mmole”represents millimole(s), “kb” represents kilobase, “bp” represents abase pair(s), and “IU” represents an international unit.

The term “polymerase chain reaction” is abbreviated as PCR; the term“reverse transcriptase polymerase chain reaction” is abbreviated asRT-PCR; the term “untranslated region” is abbreviated as UTR; the term“sodium dodecyl sulfate” is abbreviated as SDS; and the term“high-pressure liquid chromatography” is abbreviated as HPLC.

The term “amplification” of DNA as used in the present invention denotesthe use of a polymerase chain reaction (PCR) to increase a concentrationof a certain DNA sequence in a mixture of DNA sequences.

The term “polynucleotide”, “nucleotide sequence” or “base sequence” is aseries of nucleotide bases (also referred to as “nucleotides”) in anucleic acid such as DNA and RNA, and represents any chain of two ormore nucleotides. A nucleotide sequence typically carries geneticinformation, including the information used by cellular machinery toproduce proteins and enzymes. The terms include double- orsingle-stranded genomic and cDNA, RNA, any synthetic and geneticallymanipulated polynucleotides, and both sense and anti-sensepolynucleotides (although only sense stands are being represented in thepresent invention). This includes single- and double-stranded molecules,i.e., DNA-DNA, DNA-RNA, and RNA-RNA hybrids, as well as “protein nucleicacids” (PNAs) formed by conjugating bases to an amino acid backbone.This also includes nucleic acids containing modified bases, for examplethio-uracil, thio-guanine, and fluoro-uracil.

In the present invention, the nucleic acids may be flanked by naturalregulatory (expression control) sequences, or may be associated withheterologous sequences including promoters, internal ribosome entrysites (IRES) and other ribosome-binding site sequences, enhancers,response elements, suppressors, signal sequences, polyadenylationsequences, introns, 5′- and 3′-non-coding regions, and the like. Thenucleic acids may also be modified by many means known in the art.Non-limiting examples of such modifications include methylation,“capping”, substitution of one or more naturally occurring nucleotideswith an analogue, and internucleotide modifications such as, forexample, modifications with uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoroamidate, carbamate, etc.) andmodifications with charged linkages (e.g., phosphorothioate,phosphorodithioate, etc.). Polynucleotides may, for example, contain oneor more additional covalently linked moieties such as proteins (e.g.,nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.),intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals,radioactive metals, iron, oxidative metals, etc.), and alkylators. Thepolynucleotides may be derivatized by forming a methyl or ethylphosphotriester or an alkyl phosphoramidate linkage. In the presentinvention, the polynucleotides may also be modified with a label capableof providing a detectable signal either directly or indirectly.Exemplary labels include radioisotopes, fluorescent molecules, biotins,and the like.

The term “nucleic acid hybridization” refers to anti-parallel hydrogenbonding between two single-stranded nucleic acids, in which A pairs withT (or U in the case of an RNA nucleic acid) and C pairs with G. Nucleicacid molecules are “hybridizable” to each other when at least one strandof one nucleic acid molecule may form hydrogen bonds with complementarybases of another nucleic acid molecule under given stringencyconditions. The stringency of hybridization is determined, for example,by (i) a temperature at which hybridization and/or washing is performed,and (ii) an ionic strength, and (iii) a concentration of a denaturantsuch as formamide in hybridization and washing solutions, as well asother parameters. The hybridization requires that the two strandscontain substantially complementary sequences. However, some degree ofmismatches may be tolerated depending on the stringency ofhybridization. Under “low stringency” conditions, a higher percentage ofmismatches are tolerable (i.e., will not prevent formation of ananti-parallel hybrid).

Typically, hybridization of two strands with high stringency requiresthat the two strands have sequences exhibiting a high degree ofcomplementarity over extended portions of lengths thereof. Examples ofhigh stringency conditions include: hybridization to filter-bound DNA in0.5 M NaHPO₄, 7% SDS, and 1 mM EDTA at 65° C., followed by washing in0.1×SSC/0.1% SDS at 68° C. (where 1×SSC includes 0.15 M NaCl and 0.15 Msodium citrate) or washing for oligonucleotide molecules in 6×SSC/0.5%sodium pyrophosphate at approximately 37° C. (in the case ofapproximately 14 nucleotide-long oligos), at approximately 48° C. (inthe case of approximately 17 nucleotide-long oligos), at approximately55° C. (in the case of 20 nucleotide-long oligos), and at approximately60° C. (in the case of 23 nucleotide-long oligos). Therefore, the term“high-stringency hybridization” refers to a combination of a solvent anda temperature in which two strands will pair to form a “hybrid” helixonly when the two strands have almost perfectly complementary nucleotidesequences.

Intermediate or moderate stringency conditions (for example, an aqueoussolution of 2×SSC at 65° C.; optionally, for example, hybridization tofilter-bound DNA in 0.5 M NaHPO₄, 7% SDS, and 1 mM EDTA at 65° C.,followed by washing in 0.2×SSC/0.1% SDS at 42° C.), and low stringencyconditions (for example, an aqueous solution of 2×SSC at 55° C.) requirethat two strands have the corresponding less overall complementaritynecessary for hybridization to occur between two sequences thereof.Certain temperature and salt conditions for any given stringencyhybridization reaction depend on a concentration of target DNA and alength and base compositions of a probe, and are generally determinedempirically in conventional preliminary experiments.

As used in the present invention, the term “standard hybridizationconditions” refers to a hybridization condition that allowshybridization of sequences having at least 75% sequence identity.According to specific exemplary embodiments, hybridization conditionswith higher stringency may be used to allow hybridization of onlysequences having at least 80% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, or at least 99% sequenceidentity.

A nucleic acid molecule that “hybridizes” to any desired nucleic acidsof the present invention may have any length. In one exemplaryembodiment, such nucleic acid molecule has a length of at least 10, atleast 15, at least 20, at least 30, at least 40, at least 50, and atleast 70 nucleotides. In another exemplary embodiment, a nucleic acidmolecule to be hybridized has substantially the same length as certaindesired nucleic acids.

The term “isolated” means that a target material is removed fromenvironments in which the target material is normally found. Therefore,an isolated biological material may be free of cellular components,i.e., components of the cells in which the biological material is foundor produced. For example, isolated nucleic acid molecules include PCRproducts, isolated mRNA, cDNA, or restriction fragments. For example,the isolated nucleic acid molecules also include sequences inserted intoplasmids, cosmids, artificial chromosomes, and the like. The isolatednucleic acid molecules are preferably excised from the genome in whichthe nucleic acid molecules may be found. More preferably, the isolatednucleic acid molecules are no longer joined to non-regulatory sequences,non-coding sequences, or to other genes located upstream or downstreamof the nucleic acid molecules when found in the genome. An isolatedprotein may be associated with other proteins or nucleic acids, or both,with which the isolated protein is associated in the cell, or associatedwith cellular membranes when the isolated protein is amembrane-associated protein.

The term “host cell” includes individual cells or cell culture brothswhich may be a recipient for vectors or a recipient for incorporation ofpolynucleotide molecules, or may have the recipient. In the presentinvention, a host cell may be a bacterium, a mammalian cell, an insectcell, or a yeast cell.

The term “treating” or “treatment” of a condition, disorder or symptomincludes:

(1) preventing or delaying the appearance of clinical or sub-clinicalsigns of the condition, disorder or symptom from developing in a mammalthat may be afflicted with or predisposed to the condition, disorder orsymptom but does not yet experience or express clinical or subclinicalsigns of the condition, disorder or symptom; or

(2) inhibiting the condition, disorder or symptom, that is, arresting,reducing or delaying the onset of a disease or a relapse thereof (incase of maintenance therapy) or one of clinical or sub-clinical signsthereof; or

(3) relieving a disease, that is, causing regression of the condition,disorder or symptom or one of clinical or sub-clinical signs thereof.

The benefit to a subject to be treated is either statisticallysignificant or at least perceptible to patients or medical physicians.

The “immune response” refers to the development of a cell-mediatedand/or antibody-mediated immune response to a composition or vaccine ofinterest in the host. Such a response is generally carried out on thesubject producing antibodies, B cells, helper T cells, and/or cytotoxicT cells specifically directed to an antigen or antigens included in thecomposition or vaccine of interest. The immune response may also includeregulatory T-cells whose activities are beyond those of organisms ofinterest, and thus may suppress other immune or allergic responses.

The “therapeutically effective amount” refers to an amount of acompound, adjuvant or vaccine composition which, when administered to amammal for the purpose of treating a condition, disorder or symptom, issufficient to effect such treatment. The “therapeutically effectiveamount” will vary depending on the compound, bacteria or analogue to beadministered, as well as a disease and severity thereof, and the age,weight, physical condition, and responsiveness of the mammal to betreated.

The “prophylactically effective amount” refers to an amount effective inachieving a desired prophylactic result at a desired dose and for adesired period of time. Typically, because a prophylactic dose is usedprior to a disease or at an earlier stage of the disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Although it is possible to use the composition provided in the presentinvention for the purpose of therapy, the composition may be preferablyadministered in the form of a pharmaceutical formulation, for example,an admixture with a suitable pharmaceutical excipient, diluent orcarrier selected with regard to the intended route of administration andstandard pharmaceutical practice. Therefore, according to one aspect ofthe present invention, there is provided a pharmaceutical composition orformulation including at least one active composition, or apharmaceutically acceptable derivative thereof, in association with apharmaceutically acceptable excipient, diluent and/or carrier. Theexcipient, diluent and/or carrier should be “acceptable” in terms ofbeing compatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

The composition of the invention may be formulated to be administered inany convenient manner for use in drugs for humans or vertebrates.Therefore, the scope of the present invention includes pharmaceuticalcompositions including a product of the present invention that isadapted for use in drugs for humans or vertebrates.

According to preferred exemplary embodiments, the pharmaceuticalcomposition is conveniently administered as a liquid oral formulation.Although there are no physical limitations in delivery of theformulation, oral delivery is preferred because the oral delivery iseasy and convenient and oral formulations readily accommodate anadditional mixture such as milk, yoghurt, and infant formula. Other oralformulations are well known in the related art, and include tablets,caplets, gelcaps, capsules, and medical foods. For example, the tabletsmay be made by a well-known compression technique using a wet, dry orfluidized bed granulation method.

Such oral formulations may be provided for use in a conventional mannerwith the aid of one or more suitable excipients, diluents, and carriers.Pharmaceutically acceptable excipients assist or make possible formationof a formulation for bioactive materials, and include diluents, binders,lubricants, glidants, disintegrants, coloring agents, and otheringredients. Preservatives, stabilizers, dyes and even flavoring agentsmay also be provided in the pharmaceutical composition. Examples of thepreservatives include sodium benzoate, ascorbic acid, and an ester ofp-hydroxybenzoic acid. Antioxidants and suspending agents may be alsoused. An excipient is pharmaceutically acceptable as long as theexcipient is non-toxic and well tolerated upon ingestion, and does notinterfere with absorption of bioactive materials, as well as performinga desired function of the excipient.

Acceptable excipients, diluents, and carriers for therapeutic use arewell known in the field of pharmaceuticals, and the choice ofpharmaceutical excipients, diluents, and carriers may be selected withregard to the intended route of administration and standardpharmaceutical practice.

As used in the present invention, the phrase “pharmaceuticallyacceptable” refers to a molecular entity and composition that aregenerally regarded as physiologically tolerable.

The term “patient,” “target” or “subject” refers to a mammal andincludes human and veterinary targets.

The dosage of an adjuvant formulation or vaccine composition containingthe adjuvant will vary widely, depending upon the nature of the disease,the patient's medical history, the frequency of administration, theadministration mode, the clearance of the agents from the host, and thelike. The initial dose may be larger, followed by smaller maintenancedoses. The dose may be administered as infrequently as monthly orannually to maintain an effective immunological memory.

The term “carrier” refers to a diluent, adjuvant, excipient or vehiclewith which the compound is administered. Such a pharmaceutical carriermay be a sterile liquid such as water, and oil, including petroleum,animal oil, vegetable oil, or oil of synthetic origin, such as peanutoil, soybean oil, mineral oil, sesame oil, and the like. Water or anaqueous solution, a saline solution, and aqueous dextrose and glycerolsolutions are preferably introduced as the carrier, particularly acarrier for injectable solutions. Optionally, the carrier may be acarrier for solid formulations, including one or more selected from abinder (in the case of a compressed pill), a glidant, an encapsulatingagent, a flavoring agent, and a coloring agent, but the presentinvention is not limited thereto.

Also, the present invention encompasses a pharmaceutical composition anda vaccine. The pharmaceutical composition and vaccine composition of thepresent invention includes a pharmaceutically acceptable carrier orexcipient along with the one or more novel Shigella antigens and one ormore adjuvants. Methods of formulating the pharmaceutical compositionand vaccine are well known to those having ordinary skill in the art.

Formulations: The vaccine compositions of the present invention mayinclude pharmaceutically acceptable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. Such compositionshave various buffer contents (for example, Tris-HCl, acetate,phosphate), and a pH and ionic strength; and include additives such assurfactants and solubilizers (for example, Tween 80, Polysorbate 80),antioxidants (for example, ascorbic acid, sodium metabisulfite),preservatives (for example, Thimersol, benzyl alcohol), and bulkingsubstances (for example, lactose, mannitol); wherein the materials areincorporated into certain preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, and the like or incorporated intoliposomes. Hylauronic acid may also be used.

Oral solid formulations are contemplated for use in the presentinvention. The solid formulations may include tablets, capsules, pills,troches, lozenges, cachets, pellets, powders, or granules. Also,liposomal or proteinoid encapsulation may be used to formulate thecompositions of the present invention. Liposomal encapsulation may beused and the liposomes may be derivatized with various polymers.Generally, the formulations will include the therapeutic agents andinert ingredients which allow for protection of the stomach againstenvironments, and release of the biologically active material in theintestine.

Also, liquid formulations for oral administration, which include apharmaceutically acceptable emulsion, a solution, a suspension, andsyrup, are contemplated for use in the present invention. In this case,the liquid formulations may contain other components including inertdiluents; an adjuvant, a wetting agent, an emulsifying agent, and asuspending agent; and a sweetening agent, a flavoring agent, a coloringagent, and a perfuming agent.

For oral formulations, the location of release of the components may bethe stomach, the small intestine (the duodenum, the jejunem, or theileum), or the large intestine. A person having ordinary skill in theart may employ formulations, which will not dissolve in the stomach butwhose materials will be released into the duodenum or elsewhere in theintestine, through the use of an enteric coating. Examples of the morecommon inert ingredients that are used as the enteric coating includecellulose acetate trimellitate (CAT), hydroxypropylmethyl cellulosephthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate(PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP),Eudragit L, Eudragit S, and Shellac. Such coatings may be used as mixedfilms.

A coating or mixture of coatings may also be used on tablets, which arenot intended to protect the stomach. This may include sugar coatings, orcoatings which make it easy to swallow the tablets. Capsules may consistof a hard shell (for example, gelatin) for delivery of dry therapeuticagents (i.e., a powder), and a soft gelatin shell may be used when thecapsules are in a liquid form. A shell material for cachets may be thickstarch or other edible papers. For pills, lozenges, molded tablets ortablet triturates, a moist massing technique may be used. Theformulation of materials for capsule administration may also be in theform of a powder, a lightly compressed plug, or even a tablet. Thetherapeutic agents may be prepared by compression.

A person having ordinary skill in the art may dilute or increase avolume of the therapeutic agent using an inert material. The diluentsmay include carbohydrates, especially mannitol, β-lactose, anhydrouslactose, cellulose, sucrose, modified dextran, and starch. Certaininorganic salts, which include calcium triphosphate, magnesiumcarbonate, and sodium chloride, may be also be used as fillers. Somecommercially available diluents include Fast-Flo, Emdex, STA-Rx 1500,Emcompress, and Avicell.

In the formulation of the therapeutic agent, disintegrants may beincorporated into solid formulations. Materials used as thedisintegrates include starch, commercially available starch-baseddisintegrants, Explotab, sodium starch glycolate, Amberlite, sodiumcarboxymethyl cellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge, andbentonite, but the present invention is not limited thereto. In thiscase, all the materials may be used. The disintegrants may also beinsoluble cationic exchange resins. Powdered gums may be used as thedisintegrants and binders, and may include powdered gums such as agar,Karaya, or tragacanth. Alginic acid and sodium salts thereof are alsouseful as the disintegrants. The binders may be taken together with thetherapeutic agent to form a hard tablet, and include naturalproduct-derived materials such as acacia, tragacanth, starch, andgelatin. Other binders include methyl cellulose (MC), ethyl cellulose(EC), and carboxymethyl cellulose (CMC). Both polyvinyl pyrrolidone(PVP) and hydroxypropylmethyl cellulose (HPMC) may be used to granulatethe peptides (or derivatives) in an alcoholic solution.

An antifrictional agent may be included in the formulation to preventsticking during a formulation process. A lubricant may be used as alayer between the peptides (or derivatives) and the die wall. In thiscase, the lubricant may include stearic acid including magnesium andcalcium salts thereof, polytetrafluoroethylene (PTFE), liquid paraffin,vegetable oil, and wax, but the present invention is not limitedthereto. Soluble lubricants such as sodium lauryl sulfate, magnesiumlauryl sulfate, polyethylene glycols having various molecular weights,and Carbowax 4000 and 6000 may also be used as the lubricant.

A glidant that may improve the fluidity of drugs during formulation andmay aid in rearranging the drugs during compression may be added. Theglidant may include starch, talc, pyrogenic silica, and hydratedsilicoaluminate.

To aid in dissolving the therapeutic agent into an aqueous environment,a surfactant might be added as a wetting agent. The surfactant mayinclude anionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents may beused, and may include benzalkonium chloride or benzethomium chloride.The list of potential nonionic detergents that may be included as thesurfactant in the formulation includes Lauromacrogol 400, polyoxyl 40stearate, polyoxyethylene-hydrogenated castor oil 10, 50 and 60,glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acidester, methyl cellulose, and carboxymethyl cellulose. Such surfactantsmay be present in the formulation of the proteins or derivatives eitheralone or in a mixture thereof in different ratios.

Controlled-release oral formulations may be used to put the presentinvention into practice. The therapeutic agent may be incorporated intoan inert matrix which permits diffusion or release of the therapeuticagent through a leaching mechanism like gum. A slowly decomposing matrixmay also be included in the formulation. Any enteric coatings also havea delayed-release effect. Other types of the controlled release arerealized by a method based on an Oros therapeutic system (Alza Corp.),that is, a method in which the therapeutic agent is enclosed in asemipermeable membrane which allows the entry of water and the releaseof agents through a single small opening due to an osmotic effect.

Other coatings may be used for the formulation. Such coatings include avariety of sugars which may be applied to coating pans. The therapeuticagent may also be provided in a film-coated tablet, and materials usedin this case are divided into two categories. The first categoryconsists of nonenteric materials, and includes methyl cellulose, ethylcellulose, hydroxyethyl cellulose, methylhydroxyethyl cellulose,hydroxypropyl cellulose, hydroxypropylmethyl cellulose, sodiumcarboxymethyl cellulose, providone, and polyethylene glycol. The secondcategory consists of enteric materials that are generally esters ofphthalic acid. A mixture of materials may be used to provide the optimumfilm coating. The film coating may be performed in a pan coater or in afluidized bed, or performed by compression coating.

In one exemplary embodiment, the Shigella strain disclosed in thepresent invention may be administered with a pharmaceutically acceptablediluent. Such formulations may be administered by injection(subcutaneous, intradermal, or intramuscular) or may be topicallyapplied onto the skin using an adhesive patch. On the other hand, thevaccine is administered via a mucosal route (oral, buccal, sublingual,nasal drops, aerosol, or rectal) using a pharmaceutically acceptablevehicle. The Shigella strain may also be mixed with an adjuvant toenhance a subsequent immune response. Example of such an adjuvantinclude an aluminium salt, ISCOM, a saponin-based adjuvant, anoil-in-water emulsion, a water-in-oil emulsion, a toll-like receptorligand such as a muramyl dipeptide, E. coli LPS, an oligonucleotidecontaining unmethylated DNA, poly(I:C), lipoteichoic acid, and apeptidoglycan, but the present invention is not limited thereto.Enterotoxins and adjuvants thereof include active derivatives such ascholera toxins, heat-labile E. coli enterotoxins, pertussis toxins,Shiga toxins, and analogues.

Preparations for parenteral administration according to the presentinvention include a sterile aqueous or non-aqueous solution, asuspension, or an emulsion. Examples of non-aqueous solvents or vehiclesinclude propylene glycol, polyethylene glycol, vegetable oils, such asolive oil and corn oil, gelatin, and injectable organic esters such asethyl oleate. Such formulations may also contain an adjuvant, apreservative, a wetting agent, an emulsifier, and a dispersing agent.The pharmaceutical compositions may, for example, be sterilized byfiltering the compositions through a bacteria-retaining filter,incorporating a sterilizing agent into the compositions, irradiating thecompositions, or heating the compositions. These compositions may alsobe prepared using sterile water or other sterile injectable media,immediately before use thereof.

Vaccines: In the case of vaccines, it is often observed that a primarychallenge with an antigen alone, in the absence of an adjuvant, fails toelicit a humoral or cellular immune response. Therefore, the vaccines ofthe invention may contain adjuvants including, but not limited to,cholera toxins, fragments and mutants or derivatives having adjuvantproperties, E. coli heat-labile enterotoxins, fragments and mutants orderivatives having adjuvant properties, oil-in-water and water-in-oilemulsions, toll-like receptor ligands such as a muramyl dipeptide, E.coli LPS, oligonucleotides containing unmethylated DNA, poly(I:C),lipoteichoic acid, peptidoglycans. Enterotoxins and adjuvants thereofinclude active derivatives such as cholera toxins, heat-labile E. colienterotoxins, pertussis toxins, Shiga toxins, and analogues. Otheradjuvants such as complete Freund's adjuvants, incomplete Freund'sadjuvants, saponin, mineral gels such as aluminum hydroxide, surfaceactive materials such as lysolecithin, pluronic polyols, polyanions,peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanin, andpotentially useful human adjuvants such asN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine,Bacille Calmette-Guerin (BCG), and Corynebacterium parvum may be used.An adjuvant may serve as a tissue depot that slowly releases theantigens and may also serve as a lymphoid system activator that enhancesan immune response in a non-specific manner. When the vaccine isintended for use in human subjects, the adjuvant should bepharmaceutically acceptable.

Administration: Such pharmaceutical compositions and vaccines may beadministered orally (in a solid or liquid phase), parenterally (byintramuscular, intraperitoneal, intravenous (IV), or subcutaneousinjection), transdermally (either passively or using ionophoresis orelectroporation), transmucosally (nasally, vaginally, rectally, orsublingually), or via an inhalation route of administration, oradministered using a bioerodible insert, and may be prepared intoformulations suitable for each of the routes of administration.

In one preferred exemplary embodiment, the compositions or vaccines areadministered by means of pulmonary delivery. The compositions orvaccines are delivered to the lungs of a mammal during inhalation, andtraverses the epithelial lining of the lungs into the blood stream.

According to one exemplary embodiment of the present invention, a widerange of mechanical devices designed for pulmonary delivery oftherapeutic products, including, but not limited to, a nebulizer, ametered dose inhaler, and a powdered inhaler, all of which are familiarto those skilled in the art, are contemplated. Any specific examples ofcommercially available devices suitable for the practice of the presentinvention include an Ultravent nebulizer (Mallinckrodt Inc., St. Louis,Mo.); an Acorn II nebulizer (Marquest Medical Products, Englewood,Colo.); a Ventolin metered dose inhaler (Glaxo Inc., Research TrianglePark, N.C.); and a Spinhaler powdered inhaler (Fisons Corp., Bedford,Mass.). All such devices require the use of formulations suitable fordispensing the therapeutic agent. Typically, each of the formulations isspecific to the type of devices employed, and may involve the use ofappropriate propellant materials, in addition to the conventionaldiluents, adjuvants, surfactants and/or carriers useful in therapy.Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers is contemplated.

Formulations for use with a metered dose inhaler device may generallyinclude a finely divided powder containing the therapeutic agentsuspended in a propellant with the aid of a surfactant. The propellantmay include any conventional materials employed for this purpose, forexample chlorofluorocarbon, hydrochlorofluorocarbon, hydrofluorocarbon,or hydrocarbons including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beeffectively used as the surfactant.

Formulations to be dispensed from a powdered inhaler device will includea finely divided dry powder containing the therapeutic agent, and mayalso include a bulking agent, such as lactose, sorbitol, sucrose, ormannitol, in an amount which facilitates dispersal of the powder fromthe device, e.g., 50 to 90% by weight of the formulation. Thetherapeutic agent should be most advantageously prepared in the form ofparticles having an average particle size of 10 mm (or microns) or less,most preferably 0.5 to 5 mm, for the most effective delivery to thedistal lung.

Nasal delivery or other mucosal delivery of the therapeutic agent isalso contemplated. The nasal delivery allows a direct passage of thecomposition into the blood stream without any necessity for depositionof the product in the lung after the composition is administered to thenose. Formulations for nasal delivery include those with dextran orcyclodextran and saponin as adjuvants.

The compositions or vaccines of the present invention may beadministered in conjunction with one or more additional activeingredients, pharmaceutical compositions, or vaccines. The therapeuticagent of the present invention may be administered to an animal,preferably a mammal, most preferably a human.

Dosage: Following the methodologies well-established in the related art,an effective dose and toxicity of the compounds and compositions, whichare easily used in in vitro tests, are determined in preclinical studiesusing a small animal model (for example, mice or rats) in which theShigella strain or vaccine compositions have been found to betherapeutically or prophylactically effective and in which these drugsmay be administered by the same route proposed for the human clinicaltrials.

Formulations or dosage forms for use in the present invention need notcontain a therapeutically or prophylactically effective amount of thecomponents disclosed in the present invention because suchtherapeutically or prophylactically effective amount may be achieved byadministering a plurality of such formulations or dosage forms.

For any vaccine compositions used in the method of the presentinvention, the therapeutically or prophylactically effective dose may bepreferentially estimated from an animal model. A dose-response curvederived from an animal system is then used to determine testing dosesfor the initial clinical trials in humans. To determine safety for eachof the compositions, the dose and frequency of administration shouldmeet or surpass the requirements anticipated for use in the clinicaltrials.

As disclosed in the present invention, the dose of each of thecomponents in the composition of the present invention is determined toensure that the dose administered continuously or intermittently doesnot exceed an amount determined after consideration of the results ofanimal tests and the individual symptom of a patient. Of course, thecertain dose varies depending on the dosage procedure, the symptoms of apatient or a target animal, such as age, body weight, sex, sensitivity,feed, dosage period, drugs used in combination, and the severity of adisease. The appropriate dose and dosage time under certain conditionsmay be determined by the tests based on the aforementioned indices, butmay be refined and ultimately decided based on the judgment of thepractitioner and the individual patients' circumstances (age, generalcondition, severity of symptoms, sex, etc.) according to the standardclinical techniques.

The toxicity and therapeutic or prophylactic efficacy of thecompositions of the present invention may be determined according to thestandard pharmaceutical procedure in laboratory animals, for example,determined by measuring LD₅₀ (a lethal dose for 50% of the population)and ED₅₀ (a therapeutically effective dose for 50% of the population).The dose ratio between therapeutic and toxic effects is a therapeuticindex, which may then be expressed as the ratio ED₅₀/LD₅₀. Compositionsexhibiting high therapeutic indices are preferred.

The data obtained from animal studies can be used to formulate a rangeof doses for use in humans. The therapeutically effective doses inhumans preferably fall within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The doses may vary withinthis range depending on the formulations employed and the route ofadministration used. Ideally, a single dose of each drug should be useddaily.

MODE FOR INVENTION

Hereinafter, the present invention will be described in detail withreference to exemplary embodiments thereof.

However, it should be understood that the following examples are justpreferred examples for the purpose of illustration only and are notintended to limit or define the scope of the present invention.

EXAMPLE 1 Construction of wzy Knock-Out (ΔWZY) Shigella Strains

Each of the internal DNA fragments of wzy having a length ofapproximately 600 nt (from nucleotides 121 to 720 (600 nt) (SEQ IDNO: 1) in the case of Shigella flexneri 2a; from nucleotides 5,027 to5,672 (646 nt) (SEQ ID NO: 2) in the case of Shigella flexneri 6; fromnucleotides 1,936 to 2,525 (590 nt) (SEQ ID NO: 3) in the case ofShigella dysenteriae 2; from nucleotides 5,207 to 5,796 (590 nt) (SEQ IDNO: 4) in the case of Shigella sonnei 482-79 (pWR105); and fromnucleotides 9,161 to 9,799 (639 nt) (SEQ ID NO: 5) in the case ofShigella boydii 7) was amplified by PCR using forward and reverseprimers as listed in Table 1 (C. Daniels, C. Vindurampulle, R. Morona,Overexpression and topology of the Shigella flexneri O-antigenpolymerase (Rfc/Wzy), Mol Microbiol 28 (1998) 1211-1222).

TABLE 1Nucleotide sequences of primers used in PCR cloning of internal 600 ntDNA fragments of WZY SEQ ID WZY Primers Nucleotide sequences NOS. flexneri 2a Forward 5′-  8 GGCTCTAGAAGTTTTATACTTTTAATTTTTAATTTAGTT-3′ Reverse 5′-GCCGAATTCAAATAGAACGCTGCCCAATA-3′  9 S. flexneri 6Forward 5′-TCATTTTCTAGAAAAATTGCAAACGGAAT-3′ 10 Reverse5′-AAGAAGGAATTCCTCCATTTGATTTCATGATT-3′ 11 S. Forward5′-TTTTATTCTAGAGGATTCTTTCCTGCCCCATA-3′ 12 dysenteriae 2 Reverse5′-AATTTTGAATTCACATCAACTTTCATGCCACA-3′ 13 S. sonnei 482- Forward5′-GATTCTAGACGTTGAGGTTTCACGTTTCTC-3′ 14 79 (pWR105) Reverse5′-AACGAATTCCGAAGACAGCATTCGTTCAA-3′ 15 S. boydii 7 Forward5′-GGCTCTAGATCCCATTGGTTCAATTCTTT-3′ 16 Reverse5′-CCGGAATTCTTAGCTAACAAAACGTGCTCA-3′ 17

In the primer sequences, the underlined parts represent XbaI and EcoRIrestriction sites, respectively.

The PCR fragment was cloned into a pGEM-T vector system (Promega),digested with XbaI and EcoRI enzymes, and then inserted into a suicideplasmid pSW23.oriT (D. W. Kim, G. Lenzen, A. L. Page, P. Legrain, P. J.Sansonetti, C. Parsot, The Shigella flexneri effector OspG interfereswith innate immune responses by targeting ubiquitin-conjugating enzymes,Proc Natl Acad Sci USA 102 (2005) 14046-14051). An E. coli strainBW19610 (pir⁺ Amp^(s) Cm^(r)) was transformed with each recombinantplasmid pSWwzyTr using a heat shock method. The wzy fragment of therecombinant plasmid pSWwzyTr was verified by nucleotide sequencing. Theplasmid pSWwzyTr was purified from BW19610 using a Qiagen mini-prep kit,and E. coli SM10λpir (pir⁺ Tra⁺ Amp^(s) Cm^(r)) was transformed with thepurified plasmid pSWwzyTr using the heat shock method. E. coli SM10λpirwas conjugated with streptomycin-resistant Shigella and Shigellaresistant to both streptomycin and chloramphenicol resistant, and theconjugated Shigella colonies were then isolated on a CongoRed/streptomycin/chloramphenicol agar plate (D. W. Kim, et al., ProcNatl Acad Sci USA 102 (2005) 14046-14051). Thereafter, the purified LPSsderived from the wild-type Shigella and ΔWZY were analysed by 14%Tris/Tricine SDS PAGE (30 ng/lane: 16.5% gel and 100 ng/lane in the caseof Shigella flexneri 2a WT and ΔWZY) and silver staining.

As a result, it was confirmed that the ladder pattern specific to LPS(O-antigen polymerization) was observed from the wild types, but such apattern disappeared in ΔWZY (FIG. 2).

EXAMPLE 2 Culture of ΔWZY Strains

The ΔWZY strain was cultured from aliquots (frozen at −70° C. in 80%glycerin) overnight at 37° C. on a BTCS agar supplemented with 0.01%Congo red and 100 mg/mL of streptomycin (Shigella flexneri 2a ΔWZY); 20mg/mL of ampicillin and 10 mg/mL of chloramphenicol (Shigella flexneri 6ΔWZY); 50 mg/mL of streptomycin and 10 mg/mL of chloramphenicol(Shigella dysentriae 2 ΔWZY and Shigella sonnei ΔWZY); or 25 mg/mL ofstreptomycin and 10 mg/mL of chloramphenicol (Shigella boydii 7 ΔWZY) asthe antibiotic(s). One representative Congo Red-stained colony wascultured overnight at 37° C. while stirring in a BTCS broth containing100 mg/mL of streptomycin. Next morning, aliquots of ΔWZY (1 to 2%volume of a fresh medium) were added to a fresh BTCS broth containing anappropriate antibiotic(s) for each strain as described above, andcultured at 37° C. for 2 to 3 hours. After the OD was measured at 600 nm(an OD value of 0.5 was determined as an amount of 2×10⁸ cfu/mL), theΔWZY culture broth was centrifuged, and the bacterial pellets weresuspended in PBS to a concentration of 5×10⁹ cfu/mL.

EXAMPLE 3 Immunization and Challenge of Mice

40 μL of PBS containing 1×10⁸ cfu of ΔWZY was administered to Balb/cmice (female, 6 weeks old) via an intranasal route three times atintervals of two weeks. Bacteria were inactivated with 0.13%formalin/PBS (v/v) for 2 hours. SC602-immunized mice were used as thecontrol (P. J. Sansonetti, Infect Agents Dis 2 (1993) 201-206). Only PBSwas administered to the age- and gender-matched control mice. One weekafter the last immunization, the mice were challenged with infectiouslive Shigella dysenteriae 1, Shigella flexneri 2a 2457T, Shigellaflexneri 6, Shigella sonnei through the intranasal route. The strainsused for challenge were prepared and cultured in the same manner as inthe ΔWZY strain. The survival of the mice was monitored daily for 7 to10 days.

As a result, it was revealed that the intranasal administration of theΔWZY strain provided partial or complete protection against experimentalpneumonia induced by the strains belonging to the different Shigellaspecies (S. flexneri and S. dysenteriae) and the different Shigellaserotypes (S. flexneri 2a and 6). The results of the experiments aresummarized in Table 2 and shown in FIGS. 3A to 6B.

TABLE 2 Survival rates of mice immunized with ΔWZY Challenge ChallengeNo. of Survival rate strain dose/mouse Group mice (%) S. flexneri 2a 1 ×10⁷ cfu Naive n = 5 0 WZY n = 6 100 S. flexneri 6 1 × 10⁷ cfu Naive n =6 16.7 WZY n = 6 100 S. dysenteriae 5 × 10⁶ cfu Naive n = 5 0 WZY n = 580

EXAMPLE 4 Flow Cytometric Analysis of Whole Cells by Several Kinds ofPolyclonal Sera

The equivalent number (approximately 2×10⁷ cfu) of the freshly culturedShigella wild type and ΔWZY thereof were used to stain surfaces of thewhole cells with an anti-IcsP mouse polyclonal serum. The strain wasinactivated with 0.13% formalin or PBS (v/v) for 2 hours. The wholecells were washed with PBS, and stained with a diluted solution from theantigen-specific mice. After the resulting samples were washed threetimes with PBS, an anti-mouse goat IgG-RPE antibody was added to thesamples. The cells were washed with PBS, and then analyzed by flowcytometry.

Anti-serum against PSSP-1 as partial IcsP was developed by immunizingthe mice 3 to 4 times with co-administration with dmLT (E. B. Norton, etal., Clin Vaccine Immunol 18 (2011) 546-551; J. O. Kim, et al., ClinVaccine Immunol 22 (2015) 381-388). Naive mouse serum was used as thecontrol.

As a result, it was confirmed that a larger amount of IcsP was detectedin ΔWZY, compared to the wild-type Shigella, and that the outer membraneprotein, IcsP, was buried by O-polysaccharides in the wild-type strainbut easily detected on a surface of the ΔWZY due to a shorter length ofone unit O-antigen (FIGS. 7A to 7E).

All the documents cited in this specification are included in thisspecification although it seems to show that the individual documentsare specifically and separately encompassed by the citations. Theforegoing invention is provided for the purpose of clarity inunderstanding, and is partially described in detail with reference tothe examples. Therefore, it will be apparent to those skilled in the arton the basis of the contents disclosed in the present invention thatsome changes and modifications may be made to the examples withoutdeparting from the spirit and scope of the appended claims.

1. A Shigella strain in which surface exposure of a protective antigenexisting on a cellular membrane increases due to the destruction of awzy gene of Shigella species.
 2. The Shigella strain of claim 1, whereinthe wzy gene has a base sequence selected from the group consisting ofSEQ ID NOs: 1 to
 5. 3. The Shigella strain of claim 1, wherein theprotective antigen is an IcsP2 or SigA2 protein.
 4. The Shigella strainof claim 3, wherein the IcsP2 protein has an amino acid sequence setforth in SEQ ID NO:
 6. 5. The Shigella strain of claim 3, wherein theSigA2 protein has an amino acid sequence set forth in SEQ ID NO:
 7. 6.The Shigella strain of claim 1, wherein the Shigella species is selectedfrom the group consisting of Shigella dysenteriae, Shigella flexneri,Shigella boydii, and Shigella sonnei.
 7. The Shigella strain of claim 1,wherein the Shigella species is selected from the group consisting ofShigella dysenteriae type 1, Shigella dysenteriae 2, Shigella flexneri2a, Shigella flexneri 3a, Shigella flexneri 5a, Shigella flexneri 5b,Shigella flexneri 6, Shigella boydii serotype 4, Shigella boydii 7, andShigella sonnei 482-79.
 8. A vaccine composition for treating orpreventing shigellosis, comprising the Shigella strain according to anyone of claims 1 to
 7. 9. The vaccine composition of claim 8, wherein theShigella strain is selected from the group consisting of an attenuatedstrain, a live strain, and a dead strain.
 10. The vaccine composition ofclaim 8, further comprising an adjuvant.
 11. The vaccine composition ofclaim 10, wherein the adjuvant is selected from the group consisting ofan aluminum salt, an immune stimulating complex (ISCOM), a saponin-basedadjuvant, an oil-in-water emulsion, a water-in-oil emulsion, a toll-likereceptor ligand such as a muramyl dipeptide, Escherichia coli (E. coli)LPS, an oligonucleotide containing unmethylated DNA, poly(I:C),lipoteichoic acid, a peptidoglycan, a cholera toxin, a heat-labile E.coli enterotoxin, a pertussis toxin, and a Shiga toxin.
 12. The vaccinecomposition of claim 8, which is administered by injection or via amucosal route.
 13. The vaccine composition of claim 12, wherein theinjection is selected from the group consisting of subcutaneous,intradermal, and intramuscular injections.
 14. The vaccine compositionof claim 12, wherein the mucosal route is selected from the groupconsisting of oral, buccal, sublingual, intranasal and rectal.
 15. Thevaccine composition of claim 8, wherein an effective dose of the vaccinecomposition is in a range of approximately 10 μg to approximately 2 mg.